The presence of a feeder layer improves human corneal endothelial cell proliferation by altering the expression of the transcription factors Sp1 and NFI. (November 2018)

• Record Type: Journal Article
• Authors: Le-Bel, Gaëtan; Giasson, Claude J.; Deschambeault, Alexandre; Carrier, Patrick; Germain, Lucie; Guérin, Sylvain L.
• Is Part Of: Experimental eye research. Volume 176(2018)
• Page Start: 161
• Page End: 173
• Publication Date: 2018-11
• Subjects: Human corneal endothelial cells -- Feeder layer -- Transcription factor -- Sp1 -- NFI; Ophthalmology -- Periodicals; Eye -- Periodicals; Œil -- Périodiques; Ophthalmology; Periodicals; Electronic journals; 612.8405
• DOI: 10.1016/j.exer.2018.07.009
• Languages: English
• ISSNs: 0014-4835

Abstract

Abstract: Based on the use of tissue-cultured human corneal endothelial cells (HCECs), cell therapy is a very promising avenue in the treatment of corneal endothelial pathologies such as Fuchs' dystrophy, and post-surgical corneal edema. However, once in culture, HCECs rapidly lose their phenotypic and physiological characteristics, and are therefore unsuitable for the reconstruction of a functional endothelial monolayer. Expression of NFI, a transcription factor that can either function as an activator or a repressor of gene transcription, has never been examined in endothelial cells. The present study therefore aimed to determine the impact of a non-proliferating, lethally irradiated i3T3 feeder layer on the maintenance of HCEC's morphological characteristics, and both the expression and stability of Sp1 (a strong transcriptional activator) and NFI in such cells. The typical morphology of endothelial cells was best maintained when 8 × 10 3 /cm 2 HCECs were co-cultured in the presence of 2 × 10 4 cells/cm 2 i3T3. HCECs were found to express both Sp1 and NFI in vitro . Also, the presence of i3T3 led to higher levels of Sp1 and NFI in HCECs, with a concomitant increase in their DNA binding levels (assessed by electrophoretic mobility shift assays (EMSA)). Specifically, i3T3 increased the expression of the NFIA, NFIB and NFIC isoforms, without a noticeable increase in their mRNAs (as revealed by gene profiling on microarray). Gene profiling analysis also identified a few feeder layer-dependent, differentially regulated genes whose protein products may contribute to improving the properties of HCECs in culture. Therefore, co-culturing HCECs with an i3T3 feeder layer clearly improves their morphological characteristics by maintaining stable levels of Sp1 and NFI in cell culture. Highlights: HCECs better maintain a typical compact morphology when co-cultured in the presence of i3T3. HCECs expresses both the Sp1 and NFI transcription factors in vitro . The presence of i3T3 increases the expression and DNA binding properties of Sp1 and NFI. The increased expression and DNA binding of NFI is the consequence of increased expression of the NFIA, NFIB and NFIC isoforms. Several feeder-layer dependent, differentially regulated genes that may contribute at improving the properties of HCECs were identified by gene profiling analysis.